【摘要】 为构建表达鹅星状病毒基因2型(GoAstV-2)衣壳蛋白(Cap)的重组禽腺病毒4型(FAdV-4),本研究将GoAstV-2的Cap基因表达盒插入到含有FAdV-4感染性克隆p15A-cm-FAdV4-HNJZ中FAdV4基因组的1 966 bp自然缺失区，将获得的重组感染性克隆p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2利用限制性内切酶线性化后转染鸡肝癌细胞系(chicken hepatoma cell line, LMH),拯救出表达Cap蛋白的重组病毒rFAdV4-Cap/GoAstV-2。将重组病毒在LMH细胞中连续传至第15代，提取重组病毒基因组DNA,用插入位点两侧的鉴定引物进行PCR和利用抗Cap蛋白多克隆抗体进行间接免疫荧光试验和Western blot对重组病毒进行鉴定，同时对重组病毒在LMH细胞中的复制动态进行了研究。结果显示，GoAstV-2的Cap基因能够稳定存在于重组病毒rFAdV4-Cap/GoAstV-2基因组中，而且获得稳定表达；重组病毒具有良好的体外复制能力。本研究所制备的重组病毒rFAdV4-Cap/GoAstV-2为研制防控鹅FAdV-4和GoAstV-2混合感染的新型高效二联灭活疫苗奠定了基础。更多还原

【Abstract】 To construct a recombinant fowl adenovirus 4（FAdV-4） expressing the Cap protein of goose astrovirus genotype 2（GoAstV-2）,the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cmFAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line（LMH） to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rFAdV4-Cap/GoAstV-2.After15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GOAstV-2 infected LMH cells as template.LMH cells were infected with 15^th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15^th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant virus rFAdV4-Cap/GoAstV-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-infection of FAdV-4 and GoAstV-2 in goose.更多还原